The present invention relates to subculturable lymphokine-producing human T cell hybridomas and a process for the preparation thereof. More particularly, it relates to subculturable lymphokine-producing human T cell hybridomas obtained by the cell fusion of a protein synthesis inhibitor and/or an RNA synthesis inhibitor-treated human acute leukemia cells and mitogen- or antigen-activated a human T cells in the presence of a fusion accelerator, and a process for the preparation thereof. Also, the present invention relates to a process for preparing lymphokine which comprises transplanting a lymphokine-producing human T cell hybridoma to a warm-blooded animal other than human, multiplying said hybridoma in said animal and, cultivating multiplied hybridomas in a nutrient medium.
The phenomenon of cell fusion was discovered by Y. Okada using Sendai virus (HVJ) (Y. Okada, Biken J., 1: 103 (1958)). Since the discovery of Sendai virus mediated cell fusion, this method has been greatly used in the development of the field of somatic cell genetics.
In 1975, Kohler and Milstein succeeded in utilizing the cell fusion technique in the field of immunology. That is, it was first reported in G. Kohler and C. Milstein, Nature, 256: 495 (1975) that the fusion of spleen cells obtained from immunized mice with HAT (hypoxanthine-amino- pterin-thymidine)-sensitive murine myeloma cells results in the formation of hybridomas capable of permanently producing a monoclonal antigen-specific antibody.
Lymphocytes contained in human and animal immune systems are divided broadly into cells from thymus (T cells) and cells from bone marrow (B cells).
B cells are antibody-secreting cells. The cell fusion reported by G. Kohler et al. is between mouse-derived B cells and HAT-sensitive murine myeloma cells. On the other hand, T cells are formed by differentiation and maturation of stem cells from the bone marrow in the thymus. Further, T cells circulate in the blood flow through peripheral organs such as lymph nodes and the spleen.
T cells play a significant role in controlling the immune response of a living body. It is well known that the immune response-controlling function of T cells is promoted by a soluble mediator generally called a lymphokine which is released by T cells (H. G. Kunkel and F. J. Dixon, Advances in Immunology, 29: 56 (1980), Academic Press).
Various attempts have heretofore been made to cure various diseases, such as cancer, allergy, and infectious diseases, by controlling the immune response of a living body. Lymphokine which is specific to various immune cells may be used as a more effective immunotherapeutic agent and is expected to be utilized widely in the medical field as a clinical diagnostics (Bernstein, I. D., D. E. Thor, B. Zbar and H. J. Rapp: Science 172 729 (1971) and Piessens, W. F. and W. H. Churchill: J. Immunol 114 293 (1975)). Thus, lymphokine is a medically very important substance.
In accordance with conventional methods, however, it is impossible to prepare a large amount of lymphokine, and furthermore the purity of the lymphokine that has been conventionally prepared in the past is low. Thus the utilization of lymphokine in the medical field has been seriously inhibited.
Lymphokines are non-antibody protein factor groups which are produced by lymphocytes due to, for example, an antigen-specific stimulus or mitogen stimulus. Further, lymphokines are produced mainly by T cells. Typical lymphokines and their actions are shown below:
1. Lymphokines acting on macrophages PA0 2. Lymphokines acting on polymorphonuclear leucocytes PA0 3. Lymphokines acting on lymphocytes PA0 4. Lymphokines acting on other cells PA0 Step (A) PA0 Step (B) PA0 Step (C) PA0 Step (D) PA0 Step (E)
(1) Migration inhibitory factor (MIF) PA1 (2) Macrophage activating factor (MAF) PA1 (3) Monocyte-macrophage chemotactic factor (MCF) PA1 (1) Leucocyte-migration inhibitory factor (LIF) PA1 (2) Chemotactic factor PA1 (1) Interleukin II (IL-II) PA1 (1) Lymphotoxin (LT) PA1 (2) .gamma.-Interferon (IFN-.gamma.) PA1 (3) Colony stimulating factor (CSF)
Action: prevents the migration of macrophages in vitro PA2 Action: stimulates phagocytosis, the bactericidal action, etc. of macrophages PA2 Action: causes chemotaxis of monocyte macrophages PA2 Action: prevents the migration of polymorphonuclear leucocytes in vitro PA2 Action: causes chemotaxis of neutrophil, eosinophil and basophil leucocytes PA2 Action: stimulates the division and proliferation of T cells activated by an antigen or mitogen PA2 Action: damages and peels apart L cells and HeLa cells in vitro PA2 Action: interferes with virus pathogenicity PA2 Action: acts on bone marrow lymphocyte precursor cells (GFU-C), accelerating their differentiation and proliferation into granulocytes or macrophages
The activity of the above-described lymphokines is measured in vitro. It is reported, however, that there are lymphokines whose activity, as exhibited in vitro, is recognized to correspond to that as exhibited in vivo. For example, MIFs are presumed to inhibit migration of macrophages (Takeo Kuroyanagi et al: "Lymphokine" Shin Meneki Kagaku Sosho vol. 6, p. 33, Igakushoin Co., Ltd., Tokyo (1979)).
In addition, in teleangiectatic edema, caused by a chemical mediator in, for example, a tuberculin reaction, a large accumulation of macrophages is observed. This is because the macrophages, which migrate and collect as a result of MCF derived from sensitized T cells, are further fixed by MIF. This demonstrates the correlation between MCF and MIF with living body immunological defenses and thus permits efficient treatment of foreign substances by effective accumulation and activation of macrophages.
Lymphokines which can be expected to be used as medicines in the future include MAF, lymphotoxin, interleukin II, IFN-.gamma., and CSF, as well as MCF and MIF.
Typical methods of preparing lymphokines include (1) cultivating peripheral blood lymphocytes sensitized by an antigen together with the antigen (D. J. Cameron and W. H. Churchill: J. Clin. Invest. 63 977 (1979)); (2) cultivating peripheral blood lymphocytes or spleen cells together with a mitogen (Weiser, W. Y., Greineder, D. K., Remold, H. G. et al: J. Immunol., 126 1958 (1981)); and (3) establishing an antigen-specific T cell clone by the use of T cell growth factor (IL-II) and cultivating the clone (Green, J. A., S. R. Cooperband and S. Kibrick: Science 164 1415 (1969)).
Methods (1) and (2) above need a large amount of blood and enable one to prepare only a limited amount of lymphokine. Therefore, it is difficult to prepare a large amount of pure lymphokine according to methods (1) and (2). In accordance with method (3), specific lymphokines can be prepared in the presence of IL-II. However, method (3) suffers from disadvantages in that the production of lymphokines from T cells is poor and it is difficult and expensive to obtain human IL-II.
The above-described problems are encountered when using the conventional methods because lymphokine-producing T cells cannot be subcultivated and their growth is poor even if they are cultivated in the presence of a growth factor, such as IL-II. Thus, it is very difficult at the present time to produce a sufficient amount of lymphokine for clinical use.
As a means of solving the above-described problems using the cell hybridization technique, T cell hybridomas for mice have already been established (Taniguchi, M., Saito, T. and Tada, T.: Nature 278 555-558 (1979)). Therefore, it is now possible to analyze lymphokines produced by these cells. However, such lymphokines produced by murine T cell hybridomas cannot be used for human clinical purposes. From the viewpoint of human immunology and necessity of clinical application, it has been desired to establish lymphokine-producing human T cell hybridomas which are subculturable.
It is reasonable to expect that the same method as used for the murine T cell fusion can be applied to the fusion of human T cells. Indeed, there is a method where lymphokine-producing T cells (not subculturable) and HAT (hypoxanthine-aminopterin-thymidine)-sensitive T line tumor cells (subculturable) have been fused in the presence of fusion accelerator. Therefore, only those cells which could grow on a HAT medium were screened and cloned to obtain the desired lymphokine-producing T cell hybridomas (see Catherine Grillot-Courvalin et al., Nature 292: 844 (1981)).
However, a very complicated and difficult process is required for providing HAT-sensitivity to human T line tumor cells.